Completion of a Comprehensive Mouse Knockout Resource: HG-05-007

Frequently Asked Questions (FAQs)

If an applicant is proposing to generate mutants using gene traps or
transposons, most of the mutations will land in genes but some will not. Should
an applicant propose to keep and characterize all of those mutations?

Random insertional mutagenesis efforts to date suggest that a large percentage
of these insertions will be in genes. However, there may be value in some
of the other random insertions. Whether these should be saved and how they
should be saved will be a discussion for the Research Network, which involves
the PIs of the KOMP grants, NIH staff and KOMP advisors. For the purposes
of the application, the applicant may propose to keep these non-genic insertions
frozen on-site until such time as the project determines whether or not they
should be distributed by the KOMP repositories.

Do you have to use Bacterial Artificial Chromosomes (BACs)?

No. The RFA mentions the use of BACs because that system can produce mutants
very cost effectively. However, as mentioned in the RFA, an applicant can
propose using other vector systems if s/he can support that decision with
data that demonstrates the cost effectiveness of the system.

Will you accept an application that proposes to do conditional nulls?

The RFA does not address whether or not the null mutation can be constructed
using a construct that is conditional. The objective is to generate a null
mutation and if it can be done effectively and efficiently with a conditional
construct, that is acceptable. However, the applicant will need to address
any questions about whether there is a problem with toxicity or gross genomic
rearrangement, e.g. Cre recognition of endogenous lox-like sequences. Additionally,
the applicant should provide evidence that construction of the conditional
mutant is cost effective and cost competitive with generation of a non-conditional
mutant.

What number of genes should an applicant use as the total number of genes
in the genome when proposing to generate null mutations in a majority of mouse
genes?

Currently, the annotation of the C57BL/6 genome is not complete. For purposes
of consistency, 22,000 genes should be considered the total number of genes
in the mouse genome.

I want to propose to make knockouts for a particular gene family. Can
I propose to do so under this RFA?

No. The RFA calls for high throughput efforts to produce null mutants.
For gene targeting an applicant must apply to produce a minimum of 2500 null
mutations over 5 years. The genes to be mutated will be decided upon by a
committee of scientific advisors who will consider which genes have already
been knocked out and those that remain to be targeted.

The amount of funds available through the RFA may not be sufficient to
generate the number of targeted, gene trapped or transposon mutants asked
for. Does the NIH realize that this budget will be tight?

The goal of this RFA is to produce the largest number of null mutants
possible for the dollars available. NIH recognizes that the dollar figure
given in the RFA will demand applicants to propose very aggressive programs
that minimize cost while still providing a quality product. We aim to maximize
the use of NIH dollars and intend to fund a balanced program that may include
a set of applications performing different types of knockout mutations in
order to cover as much of the genome as possible with the dollars available.
Applicants should propose the best approach they can for accomplishing the
goals of maximizing the number of null mutations, cost reduction and quality.
The actual balance of the program and costs per knockout will be determined
as a result of peer review, Council discussion and staff negotiation with
the applicants before funding.

The RFA calls for a quality assessment plan. What level of quality assessment
is needed?

The applicant should propose a quality control plan that can be justified
as being robust for the production proposed and cost effective. Applicants
should also provide information about their current quality control program
within current efforts. Once the grants are awarded, the PIs within the Research
Network of grants funded under the initiative will discuss quality control
plans to ensure that they are uniform enough to produce a quality product
at all sites.